The term “autoimmune disease” refers to a group of diseases wherein the immune system mistakenly attacks cells, tissues and organs of a person's own body. Typically, autoimmune diseases involve antibody binding of the body's own components, such as common proteins and lipids. Antibodies that bind to self-compounds (or, more typically, to compounds that are so common that they are found in every organism) are referred to as autoantibodies. As an example, autoantibody binding of phospholipids and/or phospholipid-binding plasma proteins is associated with diseases such as systemic lupus erythematosus (SLE), deep vein and recurrent arterial thrombosis, pulmonary embolisms, recurrent spontaneous abortion, thrombocytopenia, chorea, epilepsy, livedo, idiopathic pulmonary hypertension, rheumatological conditions and a host of collagenous diseases. Other diseases associated with autoantibodies include multiple sclerosis, Crohn's disease, discoid lupus erythematosus, Hashimoto's thyroiditis, psoriasis, diabetes and rheumatoid arthritis. There are about 80 different autoimmune diseases, and as a group, these diseases affect millions of people.
A conventional theory regarding the etiology of autoimmune diseases has been that these diseases are caused by an overproduction of autoantibodies in the diseased individual, possibly due to an overexpression of a gene encoding such autoantibodies. According to this theory, the blood of an affected individual contains an elevated level of the particular autoantibody causing the disease, while the blood of a normal individual contains none of the autoantibody or only a trivial amount. This theory is seemingly supported by conventional assays, in which abundant autoantibodies can be detected in blood, or blood products such as plasma or serum, from subjects having an autoimmune disease, whereas only a zero or minimal amount of autoantibodies can be detected in blood or blood products from subjects that do not have an autoimmune disease.
The present invention is based on the remarkable discovery, reported herein, that blood from normal individuals in fact contains a significant number of autoantibodies, in a wide variety of types and specificities. It is possible to detect and isolate these autoantibodies from blood or a blood product of a normal individual if the blood or blood product is treated by oxidation, by, for example with an oxidizing agent or electric current, according to a method described herein. This discovery of autoantibodies in significant quantities in normal blood is previously unreported and, to the best of the inventor's knowledge, the existence of such autoantibodies in significant quantities in normal blood was completely unknown prior to the present invention.
Without being held to any particular theory, it is evident that if autoantibodies may be obtained by manipulating normal blood taken from persons who do not have any symptoms of autoimmune disease, then it must be that the immune system of normal persons routinely creates and circulates these autoantibodies, but in some form wherein they are masked or blocked, or otherwise prevented from having any harmful effects.
The discovery of autoantibodies in significant quantities in normal individuals raises the question of why the autoantibodies are not detected in a standard assay (typically based on the binding of an antibody to its corresponding antigen) and why the autoantibodies do not cause disease symptoms in normal individuals.
Based on earlier experiments described herein, a initial tentative explanation for how normal blood could contain autoantibodies without such antibodies being detected through ordinary screening procedures and without such antibodies causing disease, was that autoantibodies in normal individuals were somehow sequestered after they are produced. For example, the sequestration could be in the form of macromolecules such as a low or high-density lipoproteins (LDL, HDL) or some other type of microparticles, vesicles or micelles that could have the ability to keep autoantibodies cordoned off and separated from other components of the bloodstream. Under this theory, autoimmune disease could be triggered, not by the production of autoantibodies per se, but by the breakdown, disruption or lack of formation of the macromolecules, microparticles, vesicles or micelles sequestering the autoantibodies. This theory seemed supported by the initial experiments wherein autoantibodies were obtained from blood or serum samples after fairly drastic manipulation of the samples including shaking and heating.
In later experiments, described herein, however, it was shown that simpler methods of the invention, such as exposing blood or a blood product to an oxidizing agent or to a DC electric current, can be enough to obtain autoantibodies from normal blood, and that the process is reversible. Further, it was found that autoantibodies could be obtained by treating commercial lvlg products, which would be free of any type of macromolecular sequestering entity. Based on these experiments, a more likely theory for how normal blood could contain autoantibodies without such antibodies being detected through ordinary screening procedures and without such antibodies causing disease, is that the autoantibodies freely circulate along with other antibodies but that the antigen binding site of autoantibodies is somehow blocked or inactivated in normal individuals. Under this theory, autoimmune disease could be triggered by oxidation to unblock the antigen-binding site of autoantibodies. Further, this theory suggests a more general mechanism by which the binding specificity of certain plasma proteins may be altered.
An immediate practical use of the discovery that forms the basis of the present invention is that it allows for an almost unlimited supply of autoantibodies to be obtained, which autoantibodies can be used as standards in diagnostic kits for the laboratory diagnosis of autoimmune and other aPL-related diseases. Previously, collection of large amounts of autoantibodies for commercial use has been difficult because it was thought that the autoantibodies had to be obtained from individuals having an autoimmune disease or testing positive for autoantibodies in standard assays. The amount of such blood that can be obtained from phlebotomy of individual patients or by pooling blood from a group of patients known to test positive for autoantibodies is limited. Other methods of obtaining autoantibodies, such as screening phage libraries as described in U.S. Pat. No. 5,885,793, may be difficult and time-consuming.
Testing blood samples for the presence or absence of masked antibodies may have important diagnostic value as it might presage or predict what antibodies could appear subsequent to oxidative stress in particular individuals